Activators of endothelial nitric oxide synthase

ABSTRACT

The regulatory peptides for constitutive nitric oxide synthase enzymes, and a peptide specific to inducible nitric oxide synthase, as well as derivatives of the peptides, homologous peptides, nucleic acids encoding the peptides, derivatives, and homologous peptides, and antibodies to the peptides, derivatives, and homologous peptides, are disclosed. The peptides, derivatives, homologous peptides, antibodies, and nucleic acids, as well as peptidomimetics, can be used in methods of modulating the activity of nitric oxide synthase enzymes, and also in methods of treating diseases or conditions modulated by production of nitric oxide by nitric oxide synthases. Assays for identifying agents which modulate the activity of the nitric oxide synthase enzymes are also described.

BACKGROUND OF THE INVENTION

Nitric oxide (NO), a small molecule which is highly toxic at moderateconcentrations, is a key messenger in mammalian physiology. NO isproduced in humans by three related enzymes which comprise the nitricoxide synthase (NOS) family.

Endothelial NOS (ENOS) produces NO which controls vascular tone (henceblood pressure), dilates the airways, and controls numerous processesdependent on local dilation of blood vessels (such as gas exchange inlungs, penile erection, and renal function). Brain or neuronal NOS (BNOSor NNOS) produces NO which functions as a neurotransmitter. It controlsperistalsis in the gut, and is implicated in neural potentiation andbrain development. NNOS and ENOS are constitutive enzymes controlled byintracellular calcium and the regulatory protein calmodulin (CAM). Whenthe level of calcium in the cell rises, NNOS and ENOS bind calmodulinand are turned on to start NO production.

A third, inducible NOS, immune NOS or macrophage NOS (INOS or MNOS), issynthesized by the immune system in response to an immune challenge.Upon induced expression, this enzyme is always active; it has acalmodulin binding site, but binds calmodulin tightly even at lowcalcium levels. INOS produces orders of magnitude more NO than other NOsynthases. This NO level is cytotoxic to tumor cells, bacteria, andother pathogenic organisms.

While INOS thus appears to be an important component of immune response,its activity is highly toxic as well. Excess production of NO by INOScan be responsible for toxic shock syndrome, septic shock, and killingof islet cells in diabetes. Excess NO production by INOS has also beenimplicated in a wide range of other autoimmune conditions, includingarthritis and other inflammatory conditions.

Thus, it is of critical importance to learn to control NO synthesis byone NOS, without interfering with the activity of other NO synthases.Currently, inhibitors of INOS also inhibit NNOS and ENOS.

SUMMARY OF THE INVENTION

The current invention concerns recently discovered intrinsic controlsite elements of constitutive nitric oxide synthases. These intrinsiccontrol site elements, referred to as "regulatory peptides," include theregulatory peptide of endothelial nitric oxide synthase (ENOS),MSGPYNSSPRPEQHKSYKIRFNSVSCSDPLVSSWRRKRKESSNTD (SEQ. ID. NO. 1); theregulatory peptide of neuronal nitric oxide synthase (NNOS)MRHPNSVQEERKSYKVRFNSVSSYSDSRKSSGDGPDLLRDNFE (SEQ. ID. NO. 2); apolypeptide specific to inducible nitric acid synthase (INOS), SEQUENCE(SEQ. ID. NO. 3). Based on this discovery, methods are now available toidentify agents that modulate (activate or inhibit) NOS activity, aswell as the agents themselves. Agents include agents that inhibit NOSactivity by blocking calmodulin activation of the NOS enzyme; agentsthat inhibit NOS activity by blocking electron transfer from NADPH to anactive site in NOS; agents that activate a constitutive NOS enzyme byantagonizing autoinhibition of a regulatory region of the NOS enzyme;and agents that modulate NOS activity by interacting with the regulatorypeptide or spatially adjacent control regions. The agents include thepeptides described above, as well as derivatives of these peptides, andhomologous peptides. Homologous peptides include substantially isolatedpeptides having an array of at least two positively charged amino acids,and an amino acid sequence of at least about 60% homology, or about 67%homology, or about 80% homology, or about 90% homology, to the aminoacid sequence of the ENOS regulatory peptide; peptides having an aminoacid sequence of at least about 60% homology, or about 67% homology, orabout 80% homology, or about 90% homology, to the amino acid sequence ofthe NNOS regulatory peptide; peptides having an amino acid sequence ofat least about 60% homology, or about 67% homology, or about 80%homology, or about 90% homology, to the amino acid sequence of theINOS-specific peptide; and peptides having an amino acid sequence of atleast about 60% homology, or about 67% homology, or about 80% homology,or about 90% homology, to the amino acid sequence of the negativelycharged loops of the NOS enzymes. The invention further concerns nucleicacids encoding the peptides, derivatives, and homologous peptides;fusions of peptides with proteins or other macromolecules (e.g.,polysaccharides); peptidomimetics of the peptides, derivatives, andhomologous peptides; and antibodies (either monoclonal or polyclonalantibodies, or fragments thereof) to the peptides, derivatives, andhomologous peptides.

The agents can be used to modulate the activity of NOS enzymes.Preferably, the agents modulate the activity of one NOS enzyme, but donot substantially affect the activity of the other NOS enzymes. Suchagents can be used to treat diseases or conditions mediated byproduction of nitric oxide by inducible nitric oxide synthase, such astoxic shock, septic shock, autoimmune diseases, inflammatory conditions,and diabetes. The agents can also be used to treat diseases orconditions mediated by production of nitric oxide by a constitutive NOSenzyme, such as hypertension, diabetes, or AIDS-related dementia.

Thus, as a result of the discovery described herein, it is now possible,for the first time, to design and/or isolate isoform-specificinhibitors, and also isoform-specific activators, of the different NOSisoforms. This overcomes the limitations of earlier methods, whichrestricted drug discovery to analogs of substrates, such as arginine, orcofactors, such as tetrahydrobiopterin. Furthermore, the currentdiscovery allows identification of activators of NOS enzymes, which waspreviously impossible.

BRIEF DESCRIPTION OF THE FIGURE

The FIGURE demonstrates the alignment of a selected set of NOS,cytochrome P450 reductase (CPR), and bacterial flavodoxin sequences,illustrating the conservation of regions involved in FMN binding.

DETAILED DESCRIPTION OF THE INVENTION

The current invention pertains to the discovery of the existence andidentity of regulatory peptides of constitutive nitric oxide synthase(NOS) enzymes. As described in the Examples below, Applicant hasidentified the regulatory peptide of constitutive NOS enzymes as anintrinsic polypeptide insert in the flavin mononucleotide (FMN) bindingdomain of endothelial nitric oxide synthase (ENOS),MSGPYNSSPRPEQHKSYKIRFNSVSCSDPLVSSWRRKRKESSNTD (SEQ. ID. NO. 1) and inbrain or neuronal nitric oxide synthase (NNOS)MRHPNSVQEERKSYKVRFNSVSSYSDSRKSSGDGPDLLRDNFE (SEQ. ID. NO. 2). Induciblenitric oxide synthase (INOS) lacks a similar polypeptide insert;instead, INOS has an INOS-specific region (the "INOS-specificpolypeptide"), amino acids 600-615 of INOS (SEQ. ID. NO. 3), a shortloop which is split and greatly extended by the introduction of theregulatory peptide (also referred to herein as the intrinsic peptide) inENOS and NNOS. Applicant has also identified a core region of the ENOSbinding domain: the array of positively charged amino acids, RRKRK (SEQID NO. 7), within the ENOS binding domain, alters activity of theenzyme.

As a result of the discovery, methods are now available for isolating oridentifying an entirely new class of NOS isoform-specific inhibitors oractivators. As disclosed herein, it is now possible to design and/orscreen for agents which modulate (either inhibit or activate) a specificisoform of NOS, without altering the activity of other isoforms. Theinvention includes both the methods by which agents can be isolated oridentified, as well as those agents so isolated and identified.

Agents of the invention include agents that modulate, and particularlyinhibit, nitric oxide synthase activity by, for example, blockingactivation of the NOS by calmodulin. The agents also include agents thatmodulate, and particularly activate, a constitutive NOS by, for example,antagonizing or interfering with autoinhibition of the enzyme by aregulatory region of the enzyme, as described herein. The agents alsoinclude agents which bind to sites spatially adjacent to the calmodulinbinding site of a constitutive NOS enzyme. The agents further includeagents that inhibit NOS by blocking electron transfer from NADPH to anactive site in NOS. Representative agents include certain substantiallyisolated peptides, including the regulatory peptides of ENOS and NNOS,the INOS-specific peptide, the negatively charged loops of INOS, ENOSand NNOS, as well as derivatives of the peptides and homologouspeptides. Nucleic acids encoding the peptides, derivatives andhomologous peptides are also available, as are antibodies specific tothe peptides, their derivatives and homologs. Agents which bind to thesepeptides, as well as agents that bind to the control regions of the NOSisoforms, and agents which bind to functional groups within theINOS-specific peptide, are also agents of the invention. In addition,methods of altering the activity of ENOS, NNOS, or INOS; methods oftreating diseases mediated by production of nitric oxide; methods oftreating conditions mediated by ENOS; and methods of treating conditionsmediated by NNOS, are available. The methods include use oradministration of the agents described herein.

The discussion below presents screening methods for agents that modulateNOS enzymes; screening methods for agents that inhibit INOS; screeningmethods for agents that activate constitutive NOS enzymes; preparationand design of agents that bind to control sites for NOS isoforms;peptides, probes, nucleic acids, and antibodies of the invention; anduses of agents, peptides, and antibodies of the invention, includingmethods of treatment of certain diseases or conditions associated withNO production in mammals, including humans.

General Screening Methods for Agents that Modulate NOS Activity

Based on the discovery of the regulatory regions of NOS isoforms, it isnow possible to identify agents which modulate the activity of a nitricoxide synthase. An agent which "modulates" the activity of a nitricoxide synthase, as used herein, is an agent which either increases ordecreases the activity of an NOS enzyme. Agents which increase theactivity of an NOS enzyme are those which activate or promote theactivity of the NOS enzyme. Agents which decrease the activity of an NOSenzyme are those which inactivate, interfere with, minimize or preventthe activity of the enzyme. Agents of the invention can modulate NOSenzyme activity independently of calmodulin activation of the NOSenzyme; that is, whether or not calmodulin is associated with the NOSenzyme, it is the agent, rather than the calmodulin, that activates theNOS enzyme.

Current screening strategies for agents that modulate NOS activity arelimited to methods that screen for agents that inhibit NOS enzymeactivity. Screening methods for arginine (and pterin) analog inhibitorsdepend upon assays which are designed to define the effect on activityof the occupation of substrate (arginine) and/or cofactor (e.g.,tetrahydrobiopterin). Conditions are selected to ensure that thelimitation on enzyme activity is imposed at the catalytic site, and onarginine and N-OH arginine hydroxylation per se rather than on thepreceding electron transfer reactions. A screen for an arginine analogNOS inhibitor includes arginine concentrations high enough to producegood activity for an extended time course in the absence of inhibition,but preferably not so high that strong inhibitors would be outcompetedby substrate for the binding site. All other conditions are adjusted toproduce maximal or nearly maximal activity; the pterin binding site issaturated, and excess levels of calcium and calmodulin are included sothat the arginine site occupancy fraction, and not electron transferfrom NADPH, is rate limiting. The strategy for screening for a pterinbased inhibitor is similar, except that the arginine concentration ispreferably saturating, and the enzyme is preferably pretreated withpterin analogs to be screened because of the slow "off rate" oftetrahydrobiopterin. This can be accomplished with pterin replete enzymeby a long (e.g., one or more hours) preincubation with potentialinhibitors, or with pterin free recombinant enzyme by a short (e.g.,less than one hour) preincubation followed by reconstitution withtetrahydrobiopterin.

In contrast to these methods, screens for inhibitory agents thatinteract with the newly-identified regulatory regions of the NOSisoforms differ in that conditions are preferably chosen so that therate limitation is imposed on the controlled steps, which are electrontransfer reactions in which reduction of the catalytic heme group byNADPH is mediated by FMN and FAD. Conditions are selected so that theenzyme is replete with all cofactors and arginine, oxygen and NADPH arefar above their Kd values for a sufficient time course to observe goodkinetics. For inhibitor selection, NADPH consumption can be measured, asit is easier to measure than NO or citrulline production. If NADPHconsumption is inhibited, NO and citrulline production must also beinhibited; however, the converse is not true, since under someconditions NADPH to oxygen electron transfer can be uncoupled fromelectron transfer.

The most important difference is the selection of calcium and calmodulinconcentrations so that nearly maximal activity is obtained in theabsence of the potential inhibitors, but also so that the control siteis not supersaturated with CA²⁺ --CAM. The excess levels of calcium andcalmodulin (CAM) used in standard NOS assays to ensure completeactivation are sufficient to mask the effects of fairly potent controlsite peptide inhibitors, because the binding domains of these inhibitorsoverlap that of Ca²⁺ --CAM, leading to competitive binding. Thediscovery of the regulatory regions, and the description of some of theproperties of the regulatory regions and the related control sites ofthe NOS isoforms, as described herein, make it possible to selectcriteria to identify agents which interact with the newly identifiedactive sites. Prior to the discovery of the regulatory regions of NOSenzymes, there was no theoretical basis to expect that such agentsexisted or could be isolated.

Bearing in mind these considerations, assays can be used to determinewhether an agent modulates NOS activity. A sample of the agent to betested (the "test agent") is contacted with a sample of NOS (therebygenerating a test sample, herein referred to as a "synthase sample");after incubation of the synthase sample under conditions appropriate foractivity of the enzyme, as described above, the level of NOS activity ismeasured. The level of NOS enzyme activity can be, for example, comparedto the amount of activity of a control sample of the NOS under the sameconditions but in the absence of the test agent. The level of INOSactivity, or of the constitutive NOS enzyme activity, is measured by anyone of several methods; the choice of method depends on the intent ofthe assay, as described above. If the level of activity in the synthasetest sample is different from the level of activity of a control sampleof the NOS under the same conditions but in the absence of the testagent, then the agent modulates the activity of the NOS.

Similarly, assays can be used to determine whether an agent modulatesthe activity of one NOS isoform without modulating the activity of otherNOS isoforms. For example, an agent can be assayed to test whether itmodulates the activity of INOS, without modulating the activity of aconstitutive NOS enzyme. A sample of the agent to be tested (the "testagent"), such as an antibody, peptide, or peptidomimetic describedabove, is contacted with a sample of INOS (thereby generating a testsample, herein referred to as a "inducible synthase sample"); afterincubation of the inducible synthase sample under conditions appropriatefor activity of the enzyme, the level of INOS activity is measured. Thelevel of INOS enzyme activity can be, for example, compared to theamount of activity of a control sample, of INOS under the sameconditions but in the absence of the test agent. Similarly, a sample ofthe constitutive NOS enzyme is contacted with the test agent, to form asecond test sample (herein referred to as the "constitutive synthasesample") after incubation of the constitutive synthase sample underconditions appropriate for activity of the enzyme, the level of activityin the constitutive synthase sample is measured, and compared with thelevel of activity in a sample of the same constitutive NOS enzyme, underthe same conditions, without the test agent. The level of INOS activity,or of the constitutive NOS enzyme activity, is measured by any one ofseveral methods; the choice of method depends on the intent of theassay, as described above.

If the level of activity in the inducible synthase test sample isdifferent from the level of activity of a control sample of INOS underthe same conditions but in the absence of the test agent, and if thelevel of activity in the constitutive synthase test sample isapproximately equal to the level of activity of a control sample of theconstitutive NOS under the same conditions but in the absence of thetest agent, then the agent modulates the activity of INOS, but does notmodulate the activity of the constitutive NOS. Conversely, if the levelof activity in the constitutive synthase test sample is different fromthe level of activity of a control sample of the constitutive NOS underthe same conditions but in the absence of the test agent, and if thelevel of activity in the inducible synthase test sample is approximatelyequal to the level of activity of a control sample of INOS under thesame conditions but in the absence of the test agent, then the agentmodulates the activity of the constitutive NOS enzyme, and does notmodulate the activity of INOS.

The selection of inhibitors with isoform specific properties involvescomparison of inhibitory effects among assays involving all threeisoforms. Such assays are performed as described above, using a test anda control sample for each NOS enzyme. In selecting for NNOS or ENOSspecific inhibitors, screens devised as described above are set up forNNOS and ENOS with the aim of selecting compounds which aresignificantly more potent inhibitors of one isoform than the other. Anagent that "specifically inhibits" a particular NOS enzyme is an agentthat inhibits that NOS isoform activity by approximately tenfold.

Screening Methods for Agents that Specifically Inhibit INOS

Because INOS lacks an analog of the autoinhibitory element of ENOS andNNOS, and is much stronger binder of CAM; INOS binding of CAM is almostirreversible. Thus, during assays to identify agents that modulate INOSactivity, excess CAM is not added: because in order to express activeINOS, CAM must be coexpressed, and INOS is isolated replete with its CAMcomplement. INOS retains a site of interaction for inhibitory peptides,but because of the effectively zero off rate for CAM these peptides mustbind to the INOS-CAM complex. Screens set up for assays of INOS whichare analogous to the ENOS and NNOS assays mentioned above, except forthe omission of added calcium and CAM, detect inhibitors which can forma ternary complex with INOS and CAM but not inhibitors which arestrictly competitive with CAM. Strategies for an INOS screen which woulddetect inhibitors competitive with CAM include co-expression of a seriesof peptide inhibitors with INOS and CAM, or production of INOS in thepresence of the inhibitor. This provides the opportunity for tightbinding inhibitors to bind to the enzyme before CAM blocked theirrecognition site.

Additional Methods for Design and/or Identification of Agents thatInhibit Specific NOS Isoforms

NOS specific inhibitors can also be identified by using neighboringstructural elements of the control site to design and screen for agentsthat interact with the control site. These elements in INOS include theshort connecting loop that is specific to inducible nitric acid synthase(SEQ ID NO. 3), which occupies the position of the extended constitutiveNOS insertion in the FMN domain, and the two flanking, negativelycharged, loop sequences which correspond to the helix-strand transitionsin INOS. An agent that binds to the loop that is specific to inducibleNOS (SEQ ID NO. 3) occupies a position in INOS that is analogous to thatof the regulatory element in constitutive NOS, and hence has potentialinhibitory character. Agents that bind to the flanking loops can havebinding domains which overlap the binding domain for the regulatoryelement, and can be inhibitory.

Similarly, alternative control site inhibitors for ENOS and NNOS havealso been identified. Agents that bind to the corresponding flankingloop sequences in those isoforms can also serve as inhibitors. Suchagents are now available. As described below, peptides corresponding toall the control site elements, both regulatory and structural, have beensynthesized; antibodies to these peptides are examples of agentsdescribed above. Other agents can be identified by using a chargecomplement strategy; by screening an appropriate peptide library usingan INOS inhibition assay; or by screening an appropriate peptide libraryusing a selection strategy involving assaying for binding to thepeptides.

Screening Methods for Agents that Activate ENOS and NNOS

Prior to the discovery of the regulatory region of the NOS isoforms, asdescribed herein, it was not known that NOS activation was possible byany means other than by CAM activation of the enzyme. Activation ofconstitutive NOS enzymes is particularly desirable, for treatment ofdiseases and conditions associated with NO production. Activation ofENOS and NNOS can most readily be visualized in terms of an agent thatbinds to the regulatory element of the isoform of interest, so as toprevent autoinhibition of NOS. An example of such an agent is anantibody to the regulatory element, which serves to interfere with theinhibited conformation. Other agents that bind to the regulatory elementcan be designed or identified by screening for activation. Design ofagents utilizes charge complementarity: since a critical feature of theregulatory elements is the presence of an array of predominantlypositive charges, activator candidates have arrays of predominantlynegative charges. Since extension of peptides containing positive chargearrays with additional residues corresponding to ENOS and NNOSregulatory elements which contain a few, or one negative charge, produceinhibitors of lower Kd than the positive charge array alone, flankingregions bearing one, or a few positive charges around a core of negativecharges may be more potent as binding agents, and hence as activators.

The flanking loop sequences of all three NOS isoforms bear negativecharges. In addition for their importance as sites for directed bindingwith regulatory potential, in constitutive NOS these sequences are partof the binding domain, and possibly part of the recognition site, of theregulatory peptide. In INOS, the corresponding sequences occupy theanalogous positions spatially adjacent to the vestigial regulatory site.Structurally, the constitutive NOS flanking loops form the edges of acup which surrounds the base of the regulatory peptide. This suggeststhat the negative charges on these loops may interact with some of thepositive charges on the regulatory peptide, and hence the syntheticpeptides derived from these loops are examples of the agents bearingnegative charge arrays discussed above. Thus, they are potentially CAMindependent activators.

Screens for CAM independent control site activation of a NOS enzyme usesassays in which conditions are selected for nearly maximum activity,except that no CAM is added. This includes saturation of the enzyme witharginine, oxygen and NADPH, and, if necessary, reconstitution withtetrahydrobiopterin. Potential activators are selected for their abilityto stimulate NO production. While it is possible to use NADPHconsumption as an initial assay for this screen, it is preferable to useNO (or citrulline) formation as a basis for the assay, as an increase inNADPH consumption could be due to an increase in the uncoupled rate ofelectron transfer to oxygen.

Design of Agents That Bind to Control Sites of NOS Isoforms

Based on the discovery of the regulatory region of the NOS isoforms,agents that bind to the NOS isoforms control sites can be developed.Initial agents that were designed and tested, as described below in theExamples, were synthetic variants of the autoinhibitory elements ofconstitutive NOS isoforms, neighboring structural elements, andcomplements (including antibodies) to these elements. The agents includepeptides which have been identified as potent inhibitors, and agentshaving the same or substantially the same characteristic properties ofthese inhibitors, including charge arrays such as the polybasicsequences found in the control elements and their complements.

Developmental strategies for agent design take several forms. Startingwith the existing inhibitory peptides, homologous or similar peptidesare sought which increase the potency of inhibition, or which improveisoform specificity. These homologous or similar sequences can beidentified either by mass screening of related sequences or bysequential variations to rationally design a superior variant.Furthermore, peptidomimetics can be devised utilizing the establishedimportance of the polybasic array in inhibitor potency. For example, apeptidomimetic can be designed by substituting the peptidic backbone ofthe molecule with an alternative oganic or inorganic backbone, wherebythe basic moieties are arranged and/or presented in the samethree-dimensional configuration. Steric hindrance can be introduced toimprove specificity and libraries of charge complemented (primarilynegative) molecules can be screened to find agents that bind to thecontrol site elements themselves.

Agents of the Invention

Agents which modulate NOS enzyme activity include agents having an arrayof positively charged residues or molecules, such as the array ofpositively charged amino acids in ENOS-homologous peptides. This agentcan modulate an NOS enzyme activity by effecting electron transportbetween NADPH and the active site of an NOS enzyme. Other agents have atleast one functional group, and may have two or more functional groups,which bind to the INOS-specific peptide; or have at least one functionalgroup, and may have two or more functional groups, which bind to therecognition site of the inhibitory peptide in a constitutive NOS enzyme,or a homologous region in inducible NOS, and thereby modulate INOSactivity. The functional group(s) of the agent can be, for example,amine groups, which bind to a carboxy group within the INOS-specificpeptide. For example, as described in Example 2 below, certain peptidesof the invention have been shown to inhibit the activity of INOS. Thesepeptides are useful as agents which modulate INOS activity.

In addition, agents that bind to neighboring structural elements canalso serve as either activators or inhibitors by interfering with CAMbinding, with the active or inactive conformation of the intrinsicautoinihibitory element, or by sterically mimicking the inhibitoryeffect of the autoinhibitory element. For example, the negativelycharged flanking loop regions form the NOS FMN binding domains areneighboring structural elements that can serve as activators orinhibitors of NOS isoforms. Representative agents include antibodies forthe synthetic peptide analogs of the NOS control elements andneighboring structural elements. For example, antibodies which areraised to the INOS-specific polypeptide (SEQ. ID. NO. 3) and/or relatedregions, can be used as agents which modulate INOS activity withoutmodulating the activity of a constitutive NOS enzyme, because theyspecifically bind to INOS, but not to a constitutive NOS enzyme.Antibodies raised to polypeptides comprising one or both of the twonegatively charged loops on the surface of the FMN binding moleculepresent in all known NOS isoforms, also modulate INOS activity withoutmodulating the activity of a constitutive NOS enzyme. The two negativelycharged loops are exposed in INOS, but are not in the constitutive NOSenzymes, because the sites appear to serve as the binding site for theregulatory peptides in ENOS and NNOS, and are therefore covered by thepositively charged regulatory peptide, or by the regulatory peptide andcalmodulin. Antibodies raised to a NOS regulatory peptide (such as SEQ.ID. NO. 1 or NO. 2), or a polypeptide comprising the NOS regulatorypeptide and/or related regions, can be used as agents which modulate theactivity of constitutive NOS enzymes without modulating the activity ofINOS, as they specifically bind to constitutive NOS enzymes, but not toINOS.

In preferred embodiments, the agent modulates the activity of INOS,without modulating the activity of a constitutive NOS enzyme (ENOSand/or NNOS); alternatively, the agent modulates the activity ofconstitutive NOS enzymes, without modulating the activity of INOS. In aneven more preferred embodiment, the agent modulates the activity of oneconstitutive NOS enzyme (ENOS or NNOS), and does not modulate theactivity of the other constitutive NOS enzyme. The agents of theinvention include peptides, as well as antibodies, and peptidomimeticswhich have the same or similar activity as the peptides, as describedbelow.

Peptides

A series of peptides has been constructed and tested for the ability tomodulate the activity of (e.g., inhibit and/or activate) the various NOSisoforms. Thus, these substantially isolated peptides relating to theregulatory peptides of NOS proteins, as well as other related peptidescan serve as agents that modulate NOS activity. A "substantiallyisolated" peptide, as described herein, refers to a peptide that hasbeen derived or removed from the environment in which it naturallyoccurs. Examples include a peptide fragment derived or removed from thenative protein, comprising or consisting essentially of the citedsequences. For example, a substantially isolated peptide would includethe cited sequence, the sequence flanked by one or more amino acidswhich are the same as or different from the amino acid sequence of thenative protein. A peptide that inhibits a NOS enzyme is referred toherein as a "nitric oxide synthase inhibitor peptide;" a peptide thatactivates a NOS enzyme is referred to herein as a "nitric oxide synthaseactivator peptide".

The peptides of the invention include the ENOS regulatory peptide,MSGPYNSSPRPEQHKSYKIRFNSVSCSDPLVSSWRRKRKESSNTD (SEQ ID NO. 1); the NNOSregulatory peptide, MRHPNSVQEERKSYKVRFNSVSSYSDSRKSSGDGPDLLRDNFE (SEQ IDNO. 2); the INOS specific peptide (SEQUENCE) (SEQ ID NO. 3); theregulatory region between about amino acids 590-650 of ENOS; theregulatory region between about amino acids 820-880 of NNOS; and thenegatively charged loops of INOS (amino acids 568-581 (SEQ ID NO. 4) and633-647 (SEQ ID NO. 5)); ENOS (amino acids 557-570 (SEQ ID NO. 6) and666-680 (SEQ ID NO. 7); and NNOS (amino acids 790-803 (SEQ ID NO. 8) and897-911 (SEQ ID NO. 9). The peptides of the invention additionallyinclude derivatives of the ENOS regulatory peptide, NNOS regulatorypeptide, INOS specific peptide, regulatory regions of ENOS and NNOS, andnegatively charged loops of INOS, ENOS and NNOS. A "derivative" of apeptide, as described herein, is a peptide which has one or more aminoacids deleted or inserted, or has one or more conservativesubstitutions. A "conservative substitution", as used herein, is thereplacement of a first amino acid with a second amino acid that issimilar to the first amino acid in charge, polarity, reactivity, and/orstructure. Conservative substitutions include amino acid substitutionswithin the following groupings: S, T, G, A, and P; L, M, I, and V; E, D,Q, and N; R, H, and K; and F, Y, and W. Fragments of these peptides canalso serve as agents that modulate NOS activity. A fragment of a peptidethat inhibits NOS activity is referred to herein as an "inhibitoryfragment;" a fragment of a peptide that activates NOS activity isreferred to herein as an "activating fragment."

The invention further encompasses peptides that are closely related tothe ENOS control region, NNOS control region, INOS specific peptide,regulatory regions of ENOS and NNOS, and negatively charged loops ofINOS, ENOS and NNOS, as shown by the degree of homology, and, in thecase of some peptides based on autoinhibitory elements, by the presenceof a core region containing an array of positively charged amino acids.These closely related peptides are referred to herein as "homologouspeptides". The core region of a peptide homologous to the ENOSregulatory peptide contains an array of positively charged amino acids.The array has at least two, and preferably at least five, positivelycharged amino acids. At least two of the positively charged amino acidsmust be contiguous. The positively charged amino acids can benaturally-occurring or synthetic. In a preferred embodiment, eachpositively charged amino acid is lysine, arginine, histidine, orornithine. In a more preferred embodiment, the core region is RRKRK (SEQID NO. 10); in another preferred embodiment, the core region is KKRKR(SEQ ID NO. 11). The homologous eptides of the invention are at least60% homologous, or about 67% homologous, or about 80% homologous, orabout 90% homologous to either a section of the ENOS regulatory peptide(SEQ ID NO. 1), the NNOS regulatory peptide (SEQ ID NO. 2), or the INOSspecific peptide (SEQ ID NO. 3). The percent of homology indicates theamount of identity (or similarity) between the amino acid sequences oftwo peptides. The percent similarity takes into consideration close orconservative substitutions in amino acids. In a particularly preferredembodiment, the sequence of the homologous peptide is substantially thesame as the identified sequence.

In one embodiment, a homologous peptide of the invention is asubstantially isolated peptide having an array of positively chargedamino acids, and an amino acid sequence of at least about 60% homology,or about 67% homology, or about 80% homology, or about 90% homology, oris substantially the same as, the amino acid sequence of the ENOScontrol region (SEQ ID NO. 1). In a preferred embodiment, thesubstantially isolated peptide binds to a site spatially adjacent to thecalmodulin binding site of ENOS. Particular peptides include WRRKRK (SEQID NO. 12); SSWRRKRKESS (SEQ ID NO. 13);SSPRPEQHKSYKIRFNSVSCSDPLVSSWRRKRKESS (SEQ ID NO. 14); andQHKSYKIRFNSVSCSDPLVSSWRRKRKE (SEQ ID NO. 15).

In another embodiment, a homologous peptide of the invention is asubstantially isolated peptide having an amino acid sequence of at leastabout 60% homology, or about 67% homology, or about 80% homology, orabout 90% homology to, or substantially the same as, the amino acidsequence of the NNOS control region (SEQ ID NO. 2). In a preferredembodiment, the substantially isolated peptide binds to a site that isspatially adjacent to the calmodulin binding site of NNOS. Particularpeptides include QEERKSYKVRF (SEQ ID NO. 16), RPEQHKSYKIRF (SEQ ID NO.17), SDSRKSSGDGPDLR (SEQ ID NO. 18), and QEERKSYKVRFNSVSSYSDSQKSSGDGPDL(SEQ ID NO. 19).

In further embodiments, other homologous peptides include asubstantially isolated peptide having an amino acid sequence of at leastabout 60% homology, or about 67% homology, or about 80% homology, orabout 90% homology to, or substantially the same as, the amino acidsequence of the INOS specific peptide (SEQ ID NO. 3); and asubstantially isolated peptide having an amino acid sequence of at leastabout 60% homology, or about 67% homology, or about 80% homology, orabout 90% homology to the negatively charged loops of the NOS enzymes(SEQ ID NO. 4 and 5 (INOS); 6 and 7 (ENOS), and 8 and 9 (NNOS)).

The peptides of the invention can be prepared using conventionalsynthetic methods. Alternatively, they can be prepared usingconventional methods of molecular genetics (see Sambrook, J. et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1989)). The peptides of the invention can also beprepared as fusion peptides with other peptide or proteins, or withother macromolecules, such as polysaccharides.

Antibodies to the Peptides

The peptides of the invention can be used to raise antibodies.Antibodies which are raised to the peptides can be either monoclonal orpolyclonal. The term "antibody", as used herein, encompasses bothpolyclonal and monoclonal antibodies, as well as mixtures of more thanone antibody (e.g., a cocktail of different types of monoclonalantibodies) reactive with a peptide of the invention. The term antibodyis further intended to encompass whole antibodies and/or biologicallyfunctional fragments thereof. Biologically functional antibody fragmentswhich can be used are those fragments sufficient for binding of theantibody fragment to the peptide. Once the antibodies are raised, theyare assessed for the ability to bind to the peptide. Conventionalmethods can be used to perform this assessment.

Monoclonal antibodies (mAb) reactive with a peptide of the invention canbe produced using somatic cell hybridization techniques (Kohler andMilstein, Nature 256: 495-497 (1975)) or other techniques. In a typicalhybridization procedure, a crude or purified NOS enzyme or a peptide ofthe invention as described above, (e.g., INOS-specific polypeptide, ENOSregulatory peptide, NNOS regulatory peptide, a homologous peptide, or aderivative peptide, as described above), can be used as the immunogen.An animal is immunized with the immunogen to obtain antibody-producingspleen cells. The species of animal immunized will vary depending on thespecificity of mAb desired. The antibody producing cell is fused with animmortalizing cell (e.g., a myeloma cell) to create a hybridoma capableof secreting antibodies to the peptide of the invention. The unfusedresidual antibody-producing cells and immortalizing cells areeliminated. Hybridomas producing desired antibodies are selected usingconventional techniques and the selected hybridomas are cloned andcultured.

Polyclonal antibodies can be prepared by immunizing an animal in asimilar fashion as described above for the production of monoclonalantibodies. The animal is maintained under conditions whereby antibodiesreactive with the peptide of the invention are produced. Blood iscollected from the animal upon reaching a desired titer of antibodies.The serum containing the polyclonal antibodies (antisera) is separatedfrom the other blood components. The polyclonal antibody-containingserum can optionally be further separated into fractions of particulartypes of antibodies (e.g., IgG, IgM).

Antibodies raised to either the regulatory peptide of a constitutive NOSenzyme (ENOS or NNOS), or to the INOS-specific polypeptide, can be usedto distinguish constitutive NOS enzymes from INOS, and vice versa. NNOScan be distinguished from ENOS and INOS, because NNOS has a long Nterminal extension that is lacking in ENOS and INOS; antibodies to theregulatory peptide or to the INOS-specific polypeptide can now be usedto distinguish ENOS from INOS. The potential for cross reactivitybetween NOS isoforms exists for antibodies to all other regions on theNOS surface except the regulatory peptide and the INOS-specificpolypeptide. Antibodies can also be used as agents which alter theactivity of a NOS protein, as described further below.

Peptidomimetics and Other Agents

Peptidomimetics (molecules which are not polypeptides, but which mimicaspects of their structures to bind to the same site), that are basedupon the above-described peptides, can also be generated. For example,polysaccharides can be prepared that have the same functional groups asthe peptides of the invention, and which interact with NOS enzymes inthe same manner. Peptidomimetics can be designed, for example, byestablishing the three dimensional structure of the peptide agent in theenvironment in which it is bound or will bind to the enzyme. Thepeptidomimetic comprises at least two components, the binding moiety ormoieities and the backbone or supporting structure.

The binding moieities are the chemical atoms or groups which will reactor complex (as informing a hydrogen or covalent bond) with the aminoacid(s) at the binding site of the enzyme. In general, the bindingmoieties in a peptidomimetic are the same as the peptide agent.Alternatively, the binding moieties can be an atom or chemical groupwhich will react with the enzyme in the same or similar manner. Examplesof binding moieties suitable for use in designing a peptidomimetic for abasic amino acid in a peptide are nitrogen containing groups, such asamines, ammoniums, guanidines and amides or phosphoniums. Examples ofbinding moieties suitable for use in designing a peptidomimetic for anacidic amino acid can be, for example, carboxyl, lower alkyl carboxylicacid ester, sulfonic acid, a lower alkyl sulfonic acid ester or aphosphorous acid or ester thereof.

The supporting structure is the chemical entity that, when bound to thebinding moiety or moieties, that provides the three dimensionalconfiguration of the peptidomimetic. The supporting structure can beorganic or inorganic. Examples of organic supporting structures includepolysaccharides, polymers or oligomers of organic synthetic polymers(such as, polyvinyl alcohol or polylactide). It is preferred that thesupporting structure possess substantially the same size and dimensionsas the peptide backbone or supporting structure. This can be determinedby calculating or measuring the size of the atoms and bonds of thepeptide and peptidomimetic. In one embodiment, the nitrogen of thepeptide bond can be substituted with oxygen or sulfur, thereby forming apolyester backbone. In another embodiment, the carbonyl can besubstituted with a sulfonyl group or sulfinyl group, thereby forming apolyamide. Reverse amides of the peptide can be made (e.g., substitutingone or more --CONH-- groups for a --NHCO-- group). In yet anotherembodiment, the peptide backbone can be substituted with a polysilanebackbone.

These compounds can be manufactured by known methods.

For example, a polyester corresponding to the peptide RRKRK can beprepared by the substituting a hydroxyl group for each correspondingamine group on the R and K amino acids, thereby preparing a hydroxyacidand sequentially esterifying the hydroxyacids, optionally blocking thebasic side chains and acids to minimize side reactions. Determining anappropriate chemical synthesis route can generally be readily identifiedupon determining the chemical structure using no more than routineskill.

In addition, large libraries of agents, such as those libraries that canbe constructed using well-known methods of combinatorial chemistry, canbe assayed for additional agents. Such agents can be isolated throughmethods described herein are considered to be equivalent to thedescribed agents, in that they interact with the same site on the NOSprotein, fulfill the same function (e.g., alter the activity of (inhibitor activate) a specific isoform), and therefore, can be used to treatone or more diseases in which the inhibition or activation of a specificNOS isoform is beneficial. This disclosure therefore encompasses suchagents as can be prepared by one skilled in the art through the use ofthe discoveries and methods described herein.

Probes of the Invention

Isolated nucleic acid probes, which optionally may encode the peptidesof the invention, as described above, can also be prepared. Nucleic acidprobes, particularly those encoding or corresponding to the regulatorypeptide of the constitutive NOS enzymes, ENOS and NNOS (SEQ. ID.

NO. 1 and NO. 2) or to the INOS-specific polypeptide (SEQ.

ID. NO. 3), can be used as markers for identifying and mapping NOSenzyme isoforms in humans and other organisms, as well as for probes forspecific NOS enzyme genes and mRNA, and for identification of otherenzymes with undiscovered but related regulatory sites. The nucleic acidprobes can comprise RNA, cDNA, or genomic DNA. The nucleic acid probessubstantially correspond to the native nucleic acids encoding orsubstantially corresponding to the regulatory peptide of theconstitutive NOS enzymes, ENOS and NNOS (SEQ. ID. NO. 1 and NO. 2), theINOS-specific polypeptide (SEQ. ID. NO. 3), or derivative or homologouspeptides. To identify NOS enzyme isoforms or related enzymes, a DNA orRNA sample (a test sample) is obtained from the organism of interest;the test sample is contacted with a nucleic acid probe, underhybridization conditions which allow hybridization of the nucleic acidprobe to homologous DNA or RNA in the test sample. Stringencyhybridization conditions can be adjusted to eliminate hybridization toextraneous sequences (see Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1989)). The presence (or absence) of hybridization is then detected,using conventional methods. The homologous DNA or RNA, if present, canbe isolated for further study.

Nucleic Acid Molecules of the Invention

Isolated nucleic acid molecules which encode the peptides of theinvention, as described above, as well as nucleic acid moleculescomplementary to the nucleic acid molecules, can also be prepared, usingstandard techniques (see Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1989)). Such isolated nucleic acid molecules can be used to preparepeptides of the invention. In addition, isolated nucleic acid moleculeswhich encode the antibodies of the invention, as described above, aswell as complementary nucleic acid molecules, can also be prepared andused to generate antibodies of the invention.

Methods of Treatment

The agents that modulate NOS enzyme activity, as described above, can beused to modulate the activity of a nitric oxide synthase enzyme in vivo.In a preferred embodiment, the agent is used to modulate the activity ofa nitric oxide synthase in a mammal, such as a human, in order to treata disease or condition associated with NO production in the mammal. Anagent which modulates the activity of INOS, or of a constitutive NOSenzyme (NNOS or ENOS), is administered to the mammal. The agent can beadministered subcutaneously, intravenously, intramuscularly,intraperitoneally, topically, orally, rectally, nasally, buccally,vaginally, intraurethrally, by inhalation spray, or via an implantedreservoir. Where the agent is a peptide, the agent can also beadministered via a gene transfer vector containing a nucleic acidencoding the peptide. Administration of the gene transfer vector leadsto expression of a nucleic acid sequence, resulting in production of thepeptide and modulation of the specific NOS isoform. A gene transfervector containing a nucleic acid encoding the peptide can also containtissue-specific promoters, as well as other elements (e.g., enhancerelements, splicing signals, termination and polyadenylation signals,viral replicons, bacterial plasmid sequences, or other vector nucleicacid sequences). Delivery of the vector can be targeted to particularregions or cell types (e.g., by the use of decorated liposomes, or byintroducing the vector in a specific region). For example,transformation of cells with a peptide that inhibits INOS activity wouldbe effective protection against NO-related islet cell destruction inthose at risk for early onset diabetes. In another example,transformation of endothelial cells with a peptide that activates ENOScan be used for long-term treatment of hypertension.

If the agent is an antibody, gene transfer methods can also be used toadminister a nucleic acid encoding the antibody or a fragment of theantibody. Administration of the vector leads to expression of thenucleic acid sequence, resulting in production of the antibody, and,ultimately, modulation of the specific NOS isoform.

The agent can be administered in dosage formulations containingconventional, non-toxic, physiologically-acceptable carriers, adjuvants,and/or vehicles. The form in which the agents are administered willdepend at least in part on the route by which they are administered.

The agent is administered in an effective amount, which is that amountnecessary to modulate the NOS enzyme. In treatment of a disease orcondition, the agent is administered in a therapeutically effectiveamount. A therapeutically effective amount is that amount necessary toreduce or eliminate symptoms associated with the disease or condition.The effective amount, or the therapeutically effective amount, will bedetermined on an individual basis, and will be based in part, onconsideration of the agent, the individual's size and gender, theseverity of the symptoms to be treated, the result sought, the disease,etc. Thus, the therapeutically effective amount can be determined by oneof ordinary skill in the art, employing such factors and using no morethan routine experimentation.

The therapeutically effective amount can be administered in a singledose, or a series of doses separated by appropriate intervals, such ashours, days, or weeks. The term "single dose," as used herein, can be asolitary dose, and can also be a sustained release dose, such as by acontrolled-release dosage formulation or a continuous infusion. Otherdrugs can also be administered in conjunction with the agent; e.g., avasopressor administered in conjunction with an INOS inhibitor forseptic shock treatment. More than one agent which modulates the activityof a NOS enzyme can be administered at the same time.

In one embodiment of the invention, an agent which modulates, andparticularly, which increases the activity of ENOS, is administered, inorder to treat a condition modulated by production of nitric oxide byendothelial nitric oxide synthase, such as hypertension,atherosclerosis, or acute asthma. In a preferred embodiment, the agentdoes not modulate the activity of NNOS or INOS. An agent which increasesENOS activity will allow NO to be produced directly adjacent to the siteof action. Since such an activator would not have to dissociate toproduce NO, it would be stable and would not need to be "used up" to beeffective. This is due to the mode of action of the agent; it turns on acatalyst, and each molecule of agent can cause the production of manymolecules of NO gradually over a long time course. In contrast, NOdonors such as nitroglycerin are limited by the number of NO groups theycontain (usually 1-3) and must be replaced as they are used up toproduce NO.

In the case of constitutive NOS enzymes (NNOS or ENOS), an agent whichactivates either ENOS and/or NNOS in the corpus cavernosa can be used asa means for treating male erectile dysfunction. In one embodiment, theagent is administered intraurethrally to limit systemic side effects.

In another embodiment, an agent which specifically inhibits NNOS can beused to prevent brain damage in conditions involving cerebralischemia/reperfusion injury, such as head trauma.

In another embodiment, an agent which modulates, and particularly, whichdecreases the activity of INOS is administered in order to treat acondition modulated by production of nitric oxide by INOS, such asseptic shock, toxic shock, autoimmune disease such as rheumatoidarthritis, inflammatory conditions such as inflammatory bowel disease,multiple sclerosis, or diabetes. In a preferred embodiment, the agentwhich modulates INOS activity does not modulate the activity of NNOS orENOS. Furthermore, INOS is important in dementia associated withAcquired Immune Deficiency Syndrome (AIDS), and may be involved in thedestruction of the immune system and general physical deteriorationproduced by the AIDS virus (Baldeway, T., et al., AIDS 10451-452 (1996);Brenner, T., et al., Brain Res. 641:51-56 (1994); Lipson, S. A., Devel.Neurosci. 94:145-151 (1996); Buck, M. et al., EMBO J. 151753-1763(1996); Cross, et al., J. Clin. Invest. 93:2684-2650 (1994); Boulleme,A. I. et al., J. Neuroimmunol. 60:117-124 (1995); Mayer, M., Nervenarzt65:819-827 (1994)). Therefore, an agent which modulates the activity ofINOS can be administered to combat dementia, immune system destruction,and/or physical deterioration in individuals infected with the AIDSvirus. Further, an agent which activates INOS can be used to treatdiseases relating to dysplasia, cancer, or infectious disease.Activation of INOS can produce cytotoxic levels of NO which would aid inthe elimination of dysplastic or cancerous tissue, or aid in the controlof infectious agents such as viruses, microbes, or other parasites.

The following examples are further illustrative of the presentinvention.

EXAMPLE 1

Identification of the Nitric Oxide Synthase Regulatory Region

A. Structure of Nitric Oxide Synthase Enzymes

All nitric oxide synthase (NOS) enzymes are modular in nature: theyconsist of a series of connected regions, each of which is closelyrelated to a small simple protein. The C-terminal region of NOS ishomologous to two flavoproteins, ferredoxin NADP reductase (a flavinadenine dinucleotide (FAD)-linked enzyme), and flavodoxin (a smallflavin mononucleotide (FMN) binding protein). The corresponding regionsin NOS function together to bring electrons into the enzyme forproduction of NO from arginine and oxygen. Binding of calmodulin by NNOSand ENOS turns on electron transfer from the flavin binding region tothe active site, which contains a heme cofactor. The calmodulin bindingsite in all three types of NOS is located close to the N-terminal edgeof the flavodoxin module.

Almost immediately (within ten residues) after the calmodulin bindingsite in each NOS there is a region with the sequence TETGKSEALA (SEQ IDNO. 20) or a close homologue. This sequence is recognized as thehomologue of closely related flavodoxin sequences such as TDTGKTEALA(SEQ ID NO. 21), which are involved in binding the flavin cofactor.These sequences form a dense hydrogen bond network with the phosphategroup of flavin mononucleotide (FMN).

B. Alignment of Sequences

The sequences of about twenty flavodoxins were aligned with thecorresponding regions of all available NOS sequences and with thesequences of NADPH cytochrome P450 reductase (NCPR). NCPR is a relatedflavoprotein which, like NOS, has both the FMN and FAD binding domains,but is unregulated and lacks a calmodulin binding site. All known NOSisoforms contain two negatively charged loops on the surface of the FMNbinding molecule. INOS itself contains a surface polypeptide not foundin either ENOS or NNOS. This surface polypeptide, having the sequence ofamino acids 600-615 of INOS (SEQ ID NO. 3), is referred to herein as the"INOS-specific polypeptide".

Computer alignment of the flavoprotein sequences failed to align oridentify the TETGKSEALA (SEQ ID NO. 20) regions because of a largeinsertion in the constitutive NOS enzymes (NNOS and ENOS), and becauseavailable programs do not make use of three dimensional structuralinformation. The TETGKSEALA (SEQ ID NO. 20) region and the previouslyrecognized conserved sequences which make up the FMN binding site werealigned by hand. The FIGURE shows an alignment of a selected set of NOS,NCPR and bacterial flavodoxin sequences. NCPR HUMAN, human NADPH P450reductase (SEQ ID NO. 29); NOSE BOVIN, bovine ENOS (SEQ ID NO. 30); NOSERAT, rat NNOS (SEQ ID NO. 31); NOSM MOUSE, mouse INOS (SEQ ID NO. 32);FLAV ECOLI, Escherichia coli flavodoxin (SEQ ID NO. 33); and FLAV DESVH,Desulfovibrio vulgaris flavodoxin (SEQ ID NO. 34). Critical regions,which were identified based on the three dimensional structures offlavodoxins that have been solved by X-ray crystallography were forcedto align.

C. Identification of Regulatory Region

The pattern of the insertions, identified by the alignment, revealed thecontrol mechanism of nitric oxide synthase. Compared to flavodoxins,which lack a calmodulin binding side, NCPR has no insertions of morethan five residues after the TETGKSEALA (SEQ ID NO. 20) region in theFMN binding domain. Immune (macrophage) NOS (INOS or MNOS), which bindscalmodulin but does not respond to calcium, also lacks an insertion inthe corresponding region. In contrast, the constitutive NOS isoforms(ENOS and NNOS), which bind calmodulin and are under calcium control,have extensive (40-45 residues) insertions within the FMN bindingdomain. This is the critical difference between the constitutive andinducible isoforms of NOS.

The insertions are about 100 residues downstream (towards the Cterminus) from the calmodulin binding site. On the publishedthree-dimensional structure of flavodoxin (Wapenpaugh, K., et al., PNAS70:3852-3860 (1973)), this insertion lies on an external loop at oneedge of the beta sheet which forms the core of the protein. The insertis spatially adjacent to the calmodulin binding site.

Since the insert is about 30% as large as a flavodoxin molecule, andcalmodulin is comparable in size to flavodoxin, it is clear that stericeffects are important. Calmodulin binding to INOS is unusually tight,partly because of the absence of interference from the insert. In ENOSand NNOS, since the insert and calmodulin cannot occupy the same space,calmodulin binding forces the insert out of the way. It is thisinteraction between calmodulin and the insert which turns the enzyme on.Thus, ligands which interact with the insert and the adjacent region ofthe FMN domain will therefore tend to force the switching mechanism intothe off state or the on state. In other calmodulin regulated systems,such ligands may produce a weakly active state in the absence ofcalmodulin; this weakly active state is resistant to further activation.The negatively charged loops on the surface of the FMN binding domainappear to serve as the binding site for the regulatory peptides in ENOSand NNOS. The regulatory polypeptide in INOS is the short loop which issplit and greatly extended by the introduction of the regulatorypeptides in ENOS and NNOS.

D. Structural Models

The availability of solved X-ray structures for flavodoxins allows us toposition the insertion in three dimensions relative to the calmodulinbinding site. Homology-based molecular models were constructed for theFMN binding domains of constitutive NOS, INOS and p450 reductase. Thesemodels were constructed using the Insight and Homology programs (Biosym,Inc.), and could be relaxed to a sterically and energetically reasonablestate using Discover software (Biosym, Inc.).

The backbones of the INOS and p450 reductase modules can be almostsuperimposed on the backbone of desulfovibrio flavodoxin, which appearsto be the closest solved structural homolog of the FMN binding modulesof the NOS isoforms. The structure, a Rossman fold motif, is afive-stranded, parallel β sheet, with the FMN binding site along oneedge. Two aromatic residues, W and Y in INOS, are in contact with theFMN ring system; the latter serves as a shielding residue.

Most of the corresponding backbone structure of ENOS can be superimposedon the structures of INOS and p450 reductase modules, but the insertionprojects from the edge of the sheet opposite the FMN binding site. Thebackbone of NNOS is similar to ENOS. Structurally, the insertioncorresponds to the replacement of a tight 5-10 residue α>β loop with anapproximately 50 residue structure which is about 1/3 the size of theentire FMN binding module. The conformation for the insertion iscurrently unknown.

The calmodulin binding site is directly adjacent to the N terminal edgeof the FMN binding domain. When calmodulin is bound, the site must be ina helical conformation, and can then be expected to extend almostdirectly away from the FMN binding domain because of steric constraints.Calmodulins (m.w. approximately 20 kD) are considerably larger than theentire FMN binding module. Although the insertion is midway through thesequence of the FMN binding module, it is immediately apparent that inthree dimensions it is directly adjacent to the CAM binding site.Clearly, CAM binding is likely to be sterically hindered by theinsertion, and probably demands that the insertion undergoconformational reorganization. It is therefore likely that the insertionhas more than one physiologically relevant conformation.

The proximity of the CAM binding site to the insertion, their probablesteric interactions, and the correlation between calcium/calmodulincontrol and the presence of the insertion strongly suggest that theinsertion functions as a control element. An attractive potential roleis that of an inhibitory polypeptide displaced by CAM binding. It woulddiffer from the inhibitory polypeptides in other systems, in that it isnot a CAM analog, but would be displaced from a neighboring site becauseof binding domain overlap.

EXAMPLE 2

Synthetic Polypeptide Effects

In order to evaluate the functional significance of the putativeinhibitory polypeptide, a series of synthetic polypeptides were designedwhich incorporated structural features of loop regions in the FMNdomain. Polypeptides corresponding to promising recognition sites suchas the RRKRK motif were synthesized in lengths ranging from six tothirty five residues, as shown in Table 1, below. Polypeptidescorresponding to both the ENOS and NNOS insertions were selected forevaluation. In addition to constructs based on the major insertions,polypeptides corresponding to the neighboring a>: loops in all threeisoforms were synthesized, because of the possibility that these loopsform a significant part of the binding site for the insertion in NOS.This possibility was suggested both by their proximity to the insertionand by their negative charge.

                                      TABLE 1                                     __________________________________________________________________________    Synthetic Peptides                                                            Peptide                                                                            Der..sup.1                                                                         Sequence              SEQ ID NO.                                    __________________________________________________________________________    B058-01                                                                            h ENOS                                                                             AVDTRLEELGGERT        21                                              B058-02 NNOS AVDTLLEELGGERT 22                                                B058-03 m INOS DIDQKLSHLGASQT 23                                              B058-04 b ENOS DDVVSLEHET 24                                                  B058-05 r NNOS DIVHLEHES 25                                                   B058-06 m INOS KASTLEEEQ 26                                                   B058-07 b ENOS WRRKRK 12                                                      B058-08 b ENOS SSWRRKRKESS 13                                                 B058-09 h NNOS QEERKSYKVRF 16                                                 B058-10 h NNOS RPEQHKSYKIRF 17                                                B058-11 r NNOS SDSRKSSGDGPDLR 18                                              JX2 b ENOS SSPRPEQHKSYKIRFNSVSCSDPLVSSWRRKRK 14                                 ESS                                                                         JX3 b ENOS QHKSYKIRFNSVSCSDPLVSSWRRKRKE 15                                    JX4 h NNOS QEERKSYKVRFNSVSSYSDSQKSSGDGPDL 19                                  PEP1  RPEQHKSYKIRF 27                                                         PEP2  QEERKSYKVRFNSVSSYSDSRKSSGDGPDL 28                                     __________________________________________________________________________     .sup.1 Derivation: h = human; m = mouse; b = bovine and r = rat.         

JX series polypeptides are approximately 30-mers derived from ENOS andNNOS inhibitory polypeptides. BO58 series are shorter (6-mers to15-mers) peptides derived from flanking loops or from shorter segmentsof inhibitory polypeptides. CAM binding data represents counts fromradiolabeled CAM trapped on a membrane by NNOS adhesion. INOS activitywas assayed by measuring oxidation of ferrous myoglobin by product NO.Mg/ml concentrations of larger JX series polypeptides were three timeshigher than others to keep molar concentrations comparable inexperiments. The results of experiments to determine the effects of thesynthetic polypeptides on NOS activity are shown in Table 2, below.

                  TABLE 2                                                         ______________________________________                                        CAM Binding and INOS Inhibition Data                                                                INOS     INOS   INOS                                       NNOS CAM Activity activity Activity                                          Peptide Binding 0.1 mg/ml 0.3 mg/ml 1.0 mg/ml                               ______________________________________                                        None    774 ± 40                                                                             14.9 ± .56                                                                            12.3 ± .19                                                                        13.0 ± .23                               B058-01 986 ± 58 15.0 ± .73                                             B058-02 953 ± 80 15.1 ± .03                                             B058-03 863 ± 19 14.4 ± .27                                             B058-04 903 ± 57 15.8 ± .73 14.9 ± .56                               B058-05 952 ± 52 14.6 ± .03                                             B058-06  951 ± 100 15.3 ± .32                                           B058-07 1749 ± 40  13.6 ± .31                                           B058-08 152 ± 26 14.5 ± .70                                             B058-09 686 ± 43 14.7 ± .28                                             B058-10 1339 ± 49  14.6 ± .02                                           B058-11 908 ± 56 14.7 ± .94                                             JX2 1356 ± 46  13.1 ± .11  7.7 ± .11 3.2 ± .11                    JX3  674 ± 100 12.9 ± .32  7.9 ± .11 2.9 ± .32                    JX4 659 ± 41 14.1 ± .57 10.4 ± .57 8.4 ± .57                    ______________________________________                                    

Several of the polypeptides based on ENOS insertions proved capable ofinhibiting INOS activity. A 50% inhibition of INOS was observed withinminutes after adding the polypeptides. Since the "off" constant of CAMis many orders of magnitude longer than this time frame, the resultsuggests that inhibition can be obtained without CAM displacement. Ifinhibition were dependent on simple competition between CAM and theinhibitor, it would be necessary to wait hours or days for CAM to falloff and be replaced by inhibitory; therefore, a ternary complex mustform at least transiently. While it is not possible currently tocompletely eliminate the possibility that CAM is rapidly displaced fromthe ternary complex, the fact that, in the absence of peptide boiling,does not completely displace CAM from INOS, suggests that the ternarycomplex is the inhibited species.

The most effective inhibitory polypeptides contain the motif RRKRK fromthe ENOS insertion. Partial inhibition of INOS could also be obtainedwith NNOS-based polypeptides.

A second experiment confirmed these results. NOS activity measurementswere performed using recombinant rat NNOS (purified from overexpressingHEK293 cells), recombinant bovine ENOS (purified from overexpressing E.coli) or native INOS (from immunostimulant-activated rat aortic smoothmuscle cells). Activity was determined with 1-20 pmole of NOS using akinetic 96-well microtiter plate assay based on the kinetics of NADPHconsumption (NNOS and ENOS) or Fe 2+-myoglobin oxidation. Radioligandbinding was performed after incubation of 1-2 pmole of NOS for 15minutes at 23° C. with either Bolton-Hunter labelled [125I]-calmodulin(1 nM) or [3H]-N-nitro-L-arginine (200 pM) and the indicated peptides.Results are shown in Table 3. Values are means±SEM of triplicatedeterminations.

                                      TABLE 3                                     __________________________________________________________________________    Effect of Peptides on NOS Activity and Ligand Binding                              μg/ml                                                                          NOS Activity (% of control)                                                                     Ligand binding to NOS                              Peptide                                                                            (μM)                                                                           NNOS  ENOS  INOS  [.sup.3 H]-NHA                                                                       [.sup.125 I]-CAM                            __________________________________________________________________________    B058-07                                                                            100 11.0 ± 3.3                                                                       24.0 ± 2.8                                                                       91.1 ± 5.7                                                                        91.7 ± 5.1                                                                       6.7 ± 3.6                                   (107.0)                                                                      B058-08 100 19.1 ± 0.9 27.7 ± 2.0 92.1 ± 1.3  120.7 ± 12.7                                        0.0 ± 0.5                                   (71.2)                                                                       B058-09 100 102.0 ± 2.1  93.6 ± 2.3 101.1 ± 3.7  118.9 ±                                          4.1 82.7 ± 1.1                              (68.1)                                                                       B058-10 100 54.5 ± 1.8 80.5 ± 2.0 99.7 ± 5.7 115.0 ± 8.6                                          24.7 ± 2.2                                  (63.0)                                                                       B058-11 100 98.4 ± 1.9 102.6 ± 4.1  99.7 ± 2.8 119.2 ± 3.4                                        89.2 ± 6.2                                  (72.0)                                                                       JX2 300 30.4 ± 1.7 57.2 ± 7.5 62.2 ± 2.8 116.3 ± 1.9 0.0                                          ± 7.4                                       (76.0)                                                                       JX3 300 28.2 ± 0.9 40.2 ± 4.0 64.3 ± 3.2 122.4 ± 1.0 0.0                                          ± 4.8                                       (87.7)                                                                       JX4 300 103.0 ± 1.3  80.2 ± 3.8 84.1 ± 3.5  95.7 ± 4.7 82.7                                       ± 1.1                                       (84.8)                                                                     __________________________________________________________________________

EXAMPLE 3

Effects on Calmodulin Binding

In addition to the inhibition of enzyme activity, it is possible tomodulate CAM binding to NOS with synthetic homologs of the control siteelements. The relationship between enzyme inhibition and CAM binding inthe presence of control site elements can be complex. In other systems,antibodies to inhibitory elements sometimes induce partial CAMindependent activation, while preventing CAM dependent activation. It isimportant to remember that CAM binding is only a means to displace theinhibitor.

The results of the experiments not only confirmed the function of themajor FMN module insertion as the inhibitory polypeptide, but suggesteda few details of the switching mechanism. While a number of thepolypeptides could modulate CAM binding, the series containing the RRKRK(SEQ ID NO:10) motive was the most instructive. The peptide with threeresidues following this motif (RRKRKEES, amino acids 4-11 of SEQ ID NO:13) was a potent CAM antagonist with ENOS and NNOS. CAM binding wasdecreased almost to background levels, with effects seen at the 10 uMlevel.

Polypeptides which terminated at the RRKRK motif, including goodinhibitors, were promoters of CAM binding. One polypeptide which had asingle amino acid after this motif had no significant effect on CAMbinding. Polypeptides based on the flanking loop regions had nosignificant effect on INOS activity, but tended to weakly promote thebinding of CAM to constitutive NOS.

EXAMPLE 4

Mechanism of NOS Control

The results presented here provide powerful evidence that the majorinsertion in the FMN binding module is the inhibitory polypeptide ofconstitutive NOS, and that its absence in INOS accounts for the lack ofsensitivity of INOS to calcium, and, in part, for its very tight bindingof CAM. It appears that INOS has developed from an ancestralconstitutive NOS-like protein by loss of the inhibitory peptide. The CAMbinding site in INOS and constitutive NOS is apparently related to asimilar basic region near the N terminal of P450 reductase, and may havedeveloped from such a region in a common ancestral protein.

The inhibition of INOS by synthetic analogs of the constitutive NOSinhibitory polypeptides is related to the ability of the syntheticpolypeptides to modulate CAM binding, but does not have the simpledirect relationship expected if the mechanism of peptide inhibitoraction was through CAM displacement. The reverse appears to be true:inhibition/activation of NOS at this site is driven by the occupancy ofkey sites by the inhibitory polypeptide, and CAM binding acts to modifythe binding of the intrinsic inhibitory segment to a site or sitesnearby on the surface of the enzyme.

It is not necessary to displace CAM in order to inhibit the enzyme atthe control site. The data suggest that the binding domain of theinhibitory peptide has several regions. There is at least onerecognition site which binds the RRKRK (SEQ ID NO:10)motif, and there isindication of a second such site which recognizes sequences such asEERKSYKVRF (amino acids 2-11 of SEQ ID NO: 16)and EQHKSYKIRF (aminoacids 3-12 of SEQ ID NO: 17) which occur in the N terminal half of theENOS and NNOS insertions; peptides which lack RRKRK (SEQ ID NO:10) butcontain these sequences can be inhibitors and/or CAM binding modulators.

Ability to bind to this recognition site does not automatically confereither inhibitory character or the ability to prevent CAM binding. It isapparent that CAM binding is strongly inhibited by peptides with a shortextension after the RRKRK (SEQ ID NO:10) motif; a three residueextension produced a peptide which reduced CAM binding to nearbackground levels, while even a single residue produced a smalldecrease. The ability of the two polypeptides which ended in the RRKRK(SEQ ID NO:10) motif to potentiate CAM binding strongly suggests that aregion of overlap between the CAM binding site and the peptide bindingsite exists, in which the overlap occurs between bound CAM and residuestowards the C terminal from RRKRK (SEQ ID NO:10). In the intrinsicpeptide other residues may contribute to the overlap, since it is bothlarger and more conformationally constrained than the synthetic analogsused here as probes.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 35                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Met Ser Gly Pro Tyr Asn Ser Ser Pro Arg Pr - #o Glu Gln His Lys        Ser                                                                             1               5   - #                10  - #                15              - - Tyr Lys Ile Arg Phe Asn Ser Val Ser Cys Se - #r Asp Pro Leu Val Ser                  20      - #            25      - #            30                   - - Ser Trp Arg Arg Lys Arg Lys Glu Ser Ser As - #n Thr Asp                          35          - #        40          - #        45                       - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Arg His Pro Asn Ser Val Gln Glu Glu Ar - #g Lys Ser Tyr Lys Val      1               5   - #                10  - #                15               - - Arg Phe Asn Ser Val Ser Ser Tyr Ser Asp Se - #r Arg Lys Ser Ser Gly                  20      - #            25      - #            30                   - - Asp Gly Pro Asp Leu Leu Arg Asp Asn Phe Gl - #u                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Lys Lys Ser Leu Phe Met Leu Arg Glu Leu As - #n His Thr Phe Arg Tyr      1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Gln Tyr Lys Ala Ser Thr Leu Glu Glu Glu Gl - #n Leu Leu Leu              1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - His Asp Ile Asp Gln Lys Leu Ser His Leu Gl - #y Ala Ser Gln Leu          1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Glu Tyr Asp Val Val Ser Leu Glu His Glu Al - #a Leu Val Leu              1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Ala Ala Val Asp Thr Arg Leu Glu Glu Leu Gl - #y Gly Glu Arg Leu          1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Glu Tyr Asp Ile Val His Leu Glu His Glu Al - #a Leu Val Leu              1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - His Ala Val Asp Thr Leu Leu Glu Glu Leu Gl - #y Gly Glu Arg Ile          1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Arg Arg Lys Arg Lys                                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Lys Lys Arg Lys Arg                                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Trp Arg Arg Lys Arg Lys                                                  1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Ser Ser Trp Arg Arg Lys Arg Lys Glu Ser Se - #r                          1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Ser Ser Pro Arg Pro Glu Gln His Lys Ser Ty - #r Lys Ile Arg Phe Asn      1               5   - #                10  - #                15               - - Ser Val Ser Cys Ser Asp Pro Leu Val Ser Se - #r Trp Arg Arg Lys Arg                  20      - #            25      - #            30                   - - Lys Glu Ser Ser                                                                  35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Gln His Lys Ser Tyr Lys Ile Arg Phe Asn Se - #r Val Ser Cys Ser Asp      1               5   - #                10  - #                15               - - Pro Leu Val Ser Ser Trp Arg Arg Lys Arg Ly - #s Glu                                  20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - Gln Glu Glu Arg Lys Ser Tyr Lys Val Arg Ph - #e                          1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - Arg Pro Glu Gln His Lys Ser Tyr Lys Ile Ar - #g Phe                      1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - Ser Asp Ser Arg Lys Ser Ser Gly Asp Gly Pr - #o Asp Leu Arg              1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - Gln Glu Glu Arg Lys Ser Tyr Lys Val Arg Ph - #e Asn Ser Val Ser Ser      1               5   - #                10  - #                15               - - Tyr Ser Asp Ser Gln Lys Ser Ser Gly Asp Gl - #y Pro Asp Leu                          20      - #            25      - #            30                   - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - Thr Glu Thr Gly Lys Ser Glu Ala Leu Ala                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - Thr Asp Thr Gly Lys Thr Glu Ala Leu Ala                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - Ala Val Asp Thr Leu Leu Glu Glu Leu Gly Gl - #y Glu Arg Thr              1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - Asp Ile Asp Gln Lys Leu Ser His Leu Gly Al - #a Ser Gln Thr              1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - Asp Asp Val Val Ser Leu Glu His Glu Thr                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - Asp Ile Val His Leu Glu His Glu Ser                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - Lys Ala Ser Thr Leu Glu Glu Glu Gln                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - - Arg Pro Glu Gln His Lys Ser Tyr Lys Ile Ar - #g Phe                      1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - - Gln Glu Glu Arg Lys Ser Tyr Lys Val Arg Ph - #e Asn Ser Val Ser Ser      1               5   - #                10  - #                15               - - Tyr Ser Asp Ser Arg Lys Ser Ser Gly Asp Gl - #y Pro Asp Leu                          20      - #            25      - #            30                   - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 135 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                              - - Glu Ser Ser Phe Val Glu Lys Met Lys Lys Th - #r Gly Arg Asn Ile Ile      1               5   - #                10  - #                15               - - Val Phe Tyr Gly Ser Gln Thr Gly Thr Ala Gl - #u Glu Phe Ala Asn Arg                  20      - #            25      - #            30                   - - Leu Ser Lys Asp Ala His Arg Tyr Gly Met Ar - #g Gly Met Ser Ala Asp              35          - #        40          - #        45                       - - Pro Glu Glu Tyr Asp Leu Ala Asp Leu Ser Se - #r Leu Pro Glu Ile Asp          50              - #    55              - #    60                           - - Asn Ala Leu Val Val Phe Cys Met Ala Thr Ty - #r Gly Glu Gly Asp Pro      65                  - #70                  - #75                  - #80        - - Thr Asp Asn Ala Gln Asp Phe Tyr Asp Trp Le - #u Gln Glu Thr Asp Val                      85  - #                90  - #                95               - - Asp Leu Ser Gly Val Lys Phe Ala Val Phe Gl - #y Leu Gly Asn Lys Thr                  100      - #           105      - #           110                  - - Tyr Glu His Phe Asn Ala Met Gly Lys Tyr Va - #l Asp Lys Arg Leu Glu              115          - #       120          - #       125                      - - Gln Leu Gly Ala Gln Arg Ile                                                  130              - #   135                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 168 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                              - - Gly Thr Leu Met Ala Lys Arg Val Lys Ala Th - #r Ile Leu Tyr Ala Ser      1               5   - #                10  - #                15               - - Glu Thr Gly Arg Ala Gln Ser Tyr Ala Gln Gl - #n Leu Gly Arg Leu Phe                  20      - #            25      - #            30                   - - Arg Lys Ala Phe Asp Pro Arg Val Leu Cys Me - #t Asp Glu Tyr Asp Val              35          - #        40          - #        45                       - - Val Ser Leu Glu His Glu Thr Leu Val Leu Va - #l Val Thr Ser Thr Phe          50              - #    55              - #    60                           - - Gly Asn Gly Asp Pro Pro Glu Asn Gly Glu Se - #r Phe Ala Ala Ala Leu      65                  - #70                  - #75                  - #80        - - Met Glu Met Ser Gly Pro Tyr Asn Ser Ser Pr - #o Arg Pro Glu Gln His                      85  - #                90  - #                95               - - Lys Ser Tyr Lys Ile Arg Phe Asn Ser Val Se - #r Cys Ser Asp Pro Leu                  100      - #           105      - #           110                  - - Val Ser Ser Trp Arg Arg Lys Arg Lys Glu Se - #r Ser Asn Thr Asp Ser              115          - #       120          - #       125                      - - Ala Gly Ala Gly Thr Leu Arg Phe Leu Cys Va - #l Phe Gly Leu Gly Ser          130              - #   135              - #   140                          - - Arg Ala Tyr Pro His Phe Cys Ala Phe Ala Al - #a Val Asp Thr Arg Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Glu Glu Leu Gly Gly Glu Arg Leu                                                          165                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 166 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                              - - Gly Gln Ala Met Ala Lys Arg Val Lys Ala Th - #r Ile Leu Tyr Ala        Thr                                                                             1               5   - #                10  - #                15              - - Glu Thr Gly Lys Ser Gln Ala Tyr Ala Lys Th - #r Leu Cys Glu Ile Phe                  20      - #            25      - #            30                   - - Lys His Ala Phe Asp Ala Lys Ala Met Ser Me - #t Glu Glu Tyr Asp Ile              35          - #        40          - #        45                       - - Val His Leu Glu His Glu Ala Leu Val Leu Va - #l Val Thr Ser Thr Phe          50              - #    55              - #    60                           - - Gly Asn Gly Asp Pro Pro Glu Asn Gly Glu Ly - #s Phe Gly Cys Ala Leu      65                  - #70                  - #75                  - #80        - - Met Glu Met Arg His Pro Asn Ser Val Gln Gl - #u Glu Arg Lys Ser Tyr                      85  - #                90  - #                95               - - Lys Val Arg Phe Asn Ser Val Ser Ser Tyr Se - #r Asp Ser Arg Lys Ser                  100      - #           105      - #           110                  - - Ser Gly Asp Gly Pro Asp Leu Arg Asp Asn Ph - #e Glu Ser Thr Gly Pro              115          - #       120          - #       125                      - - Leu Ala Asn Val Arg Phe Ser Val Phe Gly Le - #u Gly Ser Arg Ala Tyr          130              - #   135              - #   140                          - - Pro His Phe Cys Ala Phe Gly His Ala Val As - #p Thr Leu Leu Glu Glu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Leu Gly Gly Glu Arg Ile                                                                  165                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 123 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                              - - Arg Lys Val Met Ala Ser Arg Val Arg Ala Th - #r Val Leu Phe Ala        Thr                                                                             1               5   - #                10  - #                15              - - Glu Thr Gly Lys Ser Glu Ala Leu Ala Arg As - #p Leu Ala Thr Leu Phe                  20      - #            25      - #            30                   - - Ser Tyr Ala Phe Asn Thr Lys Val Val Cys Me - #t Asp Gln Tyr Lys Ala              35          - #        40          - #        45                       - - Ser Thr Leu Glu Glu Glu Gln Leu Leu Leu Va - #l Val Thr Thr Phe Gly          50              - #    55              - #    60                           - - Asn Gly Asp Cys Pro Ser Asn Gly Gln Thr Le - #u Lys Lys Ser Leu Phe      65                  - #70                  - #75                  - #80        - - Met Leu Arg Glu Leu Asn His Thr Phe Arg Ty - #r Ala Val Phe Gly Leu                      85  - #                90  - #                95               - - Gly Ser Ser Met Tyr Pro Gln Phe Cys Ala Ph - #e Ala His Asp Ile Asp                  100      - #           105      - #           110                  - - Gln Lys Leu Ser His Leu Gly Ala Ser Gln Le - #u                                  115          - #       120                                             - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 116 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                              - - Ala Ile Thr Gly Ile Phe Phe Gly Ser Asp Th - #r Gly Asn Thr Glu Asn      1               5   - #                10  - #                15               - - Ile Ala Lys Met Ile Gln Lys Gln Leu Gly Ly - #s Asp Val Ala Asp Val                  20      - #            25      - #            30                   - - His Asp Ile Ala Lys Ser Ser Lys Glu Asp Le - #u Glu Ala Tyr Asp Ile              35          - #        40          - #        45                       - - Leu Leu Leu Gly Ile Pro Thr Trp Tyr Tyr Gl - #y Glu Ala Gln Cys Asp          50              - #    55              - #    60                           - - Trp Asp Asp Phe Phe Pro Thr Leu Glu Glu Il - #e Asp Phe Asn Gly Lys      65                  - #70                  - #75                  - #80        - - Leu Val Ala Leu Phe Gly Cys Gly Asp Gln Gl - #u Asp Tyr Ala Glu Tyr                      85  - #                90  - #                95               - - Phe Cys Asp Ala Leu Gly Thr Ile Arg Asp Il - #e Ile Glu Pro Arg Gly                  100      - #           105      - #           110                  - - Ala Thr Ile Val                                                                  115                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 120 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                              - - Met Pro Lys Ala Leu Ile Val Tyr Gly Ser Th - #r Thr Gly Asn Thr Glu      1               5   - #                10  - #                15               - - Tyr Thr Ala Glu Thr Ile Ala Arg Glu Leu Al - #a Asp Ala Gly Tyr Glu                  20      - #            25      - #            30                   - - Val Asp Ser Arg Asp Ala Ala Ser Val Glu Al - #a Gly Gly Leu Phe Glu              35          - #        40          - #        45                       - - Gly Phe Asp Leu Val Leu Leu Gly Cys Ser Th - #r Trp Gly Asp Asp Ser          50              - #    55              - #    60                           - - Ile Glu Leu Gln Asp Asp Phe Ile Pro Leu Ph - #e Asp Ser Leu Glu Glu      65                  - #70                  - #75                  - #80        - - Thr Gly Ala Gln Gly Arg Lys Val Ala Cys Ph - #e Gly Cys Gly Asp Ser                      85  - #                90  - #                95               - - Ser Tyr Glu Tyr Phe Cys Gly Ala Val Asp Al - #a Ile Glu Glu Lys Leu                  100      - #           105      - #           110                  - - Lys Asn Leu Gly Ala Glu Ile Val                                                  115          - #       120                                             - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                              - - Ala Val Asp Thr Arg Leu Glu Glu Leu Gly Gl - #y Glu Arg Thr              1               5   - #                10                                    __________________________________________________________________________

What is claimed is:
 1. An extrinsic activator of endothelial nitricoxide synthase which antagonizes autoinhibition by interacting with anintrinsic peptide region of endothelial nitric oxide synthase, whereinthe region consists of amino acids 590-650 of endothelial nitric oxidesynthase (SEQ ID NO. 30, aa 78-138).
 2. An antibody, or fragmentthereof, which binds to an epitope comprising amino acids between aboutamino acids 590-650 in the regulatory region of endothelial nitric oxidesynthase (SEQ ID NO. 30, aa 78-138).
 3. The antibody of claim 2, whereinthe antibody activates endothelial nitric oxide synthase.
 4. Aconstitutive nitric oxide synthase activator peptide consisting of anamino acid sequence selected from the group consisting of: SEQ ID NO. 4,SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9,and activating fragments and derivatives of SEQ ID NO. 4, SEQ ID NO. 5,SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 in which theactivator function of the parent compound is conserved.
 5. An activatorof endothelial nitric oxide synthase which binds to amino acids in asequence selected from the group consisting of: SEQ ID NO. 21 and SEQ IDNO.
 24. 6. A constitutive nitric oxide synthase activator peptideconsisting of an amino acid sequence selected from the group consistingof: SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO.8, and SEQ ID NO. 9.